Spermatogenesis consists of developmental stages during which spermatogonial stem cells proliferate and differentiate into spermatozoa. This system provides an excellent model for studying cell differentiation due to the ability to biochemically characterize discrete spermatogenic stages. However, the mechanisms underlying male germ cell development and associated stage- and cell-specific gene expression are poorly understood, especially at the transcriptional level. Recent studies have demonstrated that the transcription factor CREMtau is critical for the final phase of sperm development, spermiogenesis. However, distinct mechanisms are required for meiosis and formation of pachytene spermatocytes and haploid round spermatids. We have been using transgenic mice to characterize the spermatogenic cell-specific proenkephalin promoter, which is expressed in a stage-dependent manner in pachytene spermatocytes and round spermatids. A 51-bp sequence located in the proximal 5' -region is critical for germ cell-specific expression, and novel repeat elements present within this region are specifically required. These repeat sequences bind to a factor termed PACH1, the activity of which is upregulated in pachytene spermatocytes. We have also found that the expression of TATA binding protein (TBP) is highly induced at the transcriptional level in pachytene spermatocytes and round spermatids. This induction may reflect specialized requirements associated with male germ cell development. The aims of this proposal are: 1) To clone and characterize the PACH1 protein from rat spermatogenic cells using expression cloning approaches; 2) to fully analyze the cis-acting elements within the proenkephalin germ line promoter, with special focus on the repeat elements and potential regulatory sites lying downstream; and 3) to isolate and initially characterize the regulation of the TATA binding protein promoter during spermatogenesis. These studies will hopefully provide important new insight into the transcriptional regulatin of meiosis andhaploid cell formation, and may ultimately be relevant to male infertility and contraception.